Project Summary/Abstract There is a fundamental gap in the understanding of how electronic nicotine delivery systems (ENDS) al- ter the adolescent brain. Adolescents are a high-risk population in regards to nicotine-containing products as prenatal or early exposure triggers significant changes in the prefrontal cortex. With the growing popularity of ENDS among American adolescents, there is a critical need to understand how ENDS devices alter neurobiol- ogy to trigger addiction to nicotine. This is especially true given that ENDS are unique from combustible ciga- rettes given the multitude of flavors and different nicotine formulations that are specific to only ENDS e-liquids. Additionally, pod-based ENDS (i.e., Juul) contain a significantly higher concentration of nicotine compared to combustible cigarettes and tank-based ENDS. Until this knowledge gap is closed, we face the risk of increased smoking initiation, decreased cessation, and a cumulative effect of a growing population of lifelong smokers in America. Our overall goal is to identify the key changes in neurobiology, specific to ENDS, in an adolescent mouse model system that regulates nicotine reward and reinforcement. To address this, we will utilize a novel contingent nicotine self-administration assay system that allows us to use the same e-liquid tanks and Pods popular with adolescent ENDS users in a mouse model system. This will provide high translational value as we can directly assess how ENDS directly alter neurobiology and neurophysiology. We hypothesize that directly linking self-administration behavior to nAChR upregulation and changes in neurophysiology will identify brain regions and cell-types that are critical for the initiation of nicotine addiction and continued reinforcement. The rationale behind this comes from the applicant’s previous success in corre- lating nicotine reward to nAChR upregulation. We will identify ENDS-specific changes in neurobiology with three specific aims. First, we will utilize e-Vape nicotine self-administration assays in mice to examine initiation and nicotine reinforcement of ENDS to examine the impact of nicotine dose, formulation, and flavors on vap- ing-related behavior. Second, we will use the brains from the first aim to examine nAChR upregulation and provide a direct link between self-administration behavior and nAChR upregulation. Third, we will examine changes in neurophysiology via electrophysiology and fast-scan cyclic voltammetry. This approach is innovative, in the applicant's opinion, because it establishes a direct correlation be- tween vaping-related self-administration and nAChR upregulation as well as changes in neurophysiology in an in vivo model and utilizes the exact same ENDS and e-liquids popular with human adolescent vapers. This is complemented by the use of a novel mouse expressing α4-mCherry and α6-GFP nAChR subunits that allow analysis of upregulation without the use of antibodies. The proposed research is signifi...